Regulation of Cellular Zinc Homeostasis

细胞锌稳态的调节

• 批准号: 7862866
• 负责人: David J Eide
• 金额: $ 17.59万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-16 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862866
• 关键词: Binding; Biological Assay; Carrier Proteins; Cell physiology; Cells; Complex; Cytosol; Endoplasmic Reticulum; Environment; Eukaryota; Eukaryotic Cell; Fingers; Fluorescence Resonance Energy Transfer; Foundations; Golgi Apparatus; Growth; Health; Homeostasis; Human; Life; Mammalian Cell; Mammals; Mediating; Metabolism; Metallothionein; Metals; Mitochondria; Modeling; Molecular; Nutrient; Organelles; Organism; Oxidative Stress; Pathway interactions; Physiology; Play; Process; Proteins; Role; Saccharomyces cerevisiae; Source; System; Testing; Yeasts; Zinc; Zinc Fingers; Zinc deficiency; base; cell growth regulation; cofactor; extracellular; in vivo; public health relevance; response; sensor; uptake; zinc-binding protein

DESCRIPTION (provided by applicant): Zinc is an essential nutrient because of the important roles this metal plays as a catalytic and structural cofactor. Several zinc-dependent proteins reside within organelles such as the endoplasmic reticulum (ER), Golgi, and mitochondria. Therefore, transporter proteins are needed to distribute zinc into intracellular compartments. Because excess zinc can be toxic, cells also require homeostatic mechanisms to control the intracellular levels of free or labile zinc in the cytosol and within organelles. Cellular zinc homeostasis is achieved by several mechanisms including the control of zinc uptake, efflux, vesicular storage, and binding by metallothionein. In this proposal, another important facet of zinc homeostasis is considered, i.e. the control of zinc levels within intracellular organelles. These processes will be studied using the yeast Saccharomyces cerevisiae as a model eukaryotic cell. Preliminary results have raised five central hypotheses that provide the foundation for the specific aims of this proposal: 1) The yeast Msc2 and Zrg17 proteins form a heteromeric complex that transports zinc into the ER. In Aim 1, the composition of this complex will be assessed and its zinc transport activity will be characterized. 2) It is proposed that zinc finger domains will make useful probes of zinc status in the ER and other compartments of living cells. In Aim 2, new in vivo zinc sensors will be developed based on fluorescence resonance energy transfer (FRET). These and other assays to be developed will be used to define the role of Msc2/Zrg17 and other transporters in maintaining ER zinc homeostasis. 3) Zinc transport into the ER via the Msc2/Zrg17 complex is regulated in response to zinc status by both transcriptional and post-translational control mechanisms. In Aim 3, the molecular mechanisms of these regulatory systems will be dissected and their roles in maintaining ER zinc status determined. 4) It is proposed that zinc transport into the ER is mediated by transporters in addition to the Msc2/Zrg17 complex. In Aim 4, other zinc transporters for this compartment will be identified. 5) Several studies of mammalian cells showed that zinc deficiency leads to increased oxidative stress, the source of which has long been a mystery. In Aim 5, the hypothesis that the oxidative stress of zinc deficiency arises from the loss of ER zinc homeostasis will be tested. These aims represent a cohesive and comprehensive analysis of zinc homeostasis in the secretory pathway of eukaryotic cells.7. PUBLIC HEALTH RELEVANCE: The processes of intracellular zinc transport and homeostasis within organelles are essential for basic cellular function, physiology, and human health. Despite this importance, however, we know little about the transporters involved and how they are regulated in response to zinc status and other factors. As a result of our proposed studies, we will obtain a fundamental understanding of zinc metabolism in the secretory pathway of all eukaryotes and generate new probes of intracellular zinc that will be useful in our studies as well as in the analysis of zinc metabolism in mammals and other organisms.

描述（由申请人提供）：锌是一种必需营养素，因为这种金属作为催化和结构辅助因子发挥着重要作用。几种锌依赖性蛋白存在于细胞器内，例如内质网 (ER)、高尔基体和线粒体。因此，需要转运蛋白将锌分配到细胞内区室中。由于过量的锌可能有毒，因此细胞还需要稳态机制来控制细胞质和细胞器内游离或不稳定锌的细胞内水平。细胞锌稳态是通过多种机制实现的，包括控制锌的摄取、流出、囊泡储存和金属硫蛋白的结合。在该提案中，考虑了锌稳态的另一个重要方面，即细胞内细胞器内锌水平的控制。这些过程将使用酿酒酵母作为模型真核细胞进行研究。初步结果提出了五个中心假设，为该提案的具体目标奠定了基础：1）酵母 Msc2 和 Zrg17 蛋白形成异聚复合物，将锌转运到 ER 中。在目标 1 中，将评估该复合物的组成并表征其锌转运活性。 2) 建议锌指结构域将成为活细胞内质网和其他区室中锌状态的有用探针。在目标 2 中，将开发基于荧光共振能量转移（FRET）的新型体内锌传感器。这些和其他待开发的检测将用于确定 Msc2/Zrg17 和其他转运蛋白在维持 ER 锌稳态中的作用。 3) 锌通过Msc2/Zrg17复合物转运至内质网，通过转录和翻译后控制机制响应锌状态进行调节。在目标 3 中，将剖析这些调节系统的分子机制，并确定它们在维持 ER 锌状态中的作用。 4) 据推测，除了Msc2/Zrg17复合物之外，锌转运到内质网还由转运蛋白介导。在目标 4 中，将确定该区室的其他锌转运蛋白。 5) 多项针对哺乳动物细胞的研究表明，缺锌会导致氧化应激增加，而氧化应激的来源长期以来一直是个谜。在目标 5 中，将检验缺锌引起的氧化应激是由于 ER 锌稳态丧失引起的假设。这些目标代表了对真核细胞分泌途径中锌稳态的连贯和全面的分析。7。公共健康相关性：细胞内锌转运和细胞器内稳态的过程对于基本细胞功能、生理学和人类健康至关重要。然而，尽管如此重要，我们对所涉及的转运蛋白以及它们如何根据锌状态和其他因素进行调节知之甚少。作为我们提出的研究的结果，我们将对所有真核生物分泌途径中的锌代谢有一个基本的了解，并产生新的细胞内锌探针，这将有助于我们的研究以及哺乳动物和其他生物体中锌代谢的分析。